The present invention relates generally to the field of refractive index based sensing devices. More particularly, the invention relates to a critical angle refractometer and method for sensing and monitoring interactions between an analyte and a binding layer.
Analysis of qualitative and quantitative aspects of interactions between analytes and various types of binding layers is important to a wide range of scientific and industrial applications. Consequently, sensors which monitor specific binding of a sample analyte to a particular type of ligands immobilized on the sensing surface have been developed. The term xe2x80x9cligandsxe2x80x9d here means a type of molecules exhibiting specific binding affinity to another type of molecules. The terms xe2x80x9cimmobilized binding layerxe2x80x9d, xe2x80x9cbinding layerxe2x80x9d, or xe2x80x9csensing layerxe2x80x9d here mean a layer formed by ligands immobilized on a sensing surface. The term xe2x80x9csensing surfacexe2x80x9d means an interface between two media, one of which is the binding layer. The term xe2x80x9ccontacting phasexe2x80x9d here means a fluid phase, which is brought in contact with the binding layer. The term xe2x80x9canalytexe2x80x9d, or xe2x80x9csample analytexe2x80x9d, here means the ligands contained in the contacting phase. An analyte in a contacting phase may or may not possess binding affinity to a particular binding layer.
For example, sensors based on the surface plasmon resonance (SPR) phenomenon are known to detect and measure changes in the refractive index of a sample analyte contacting a sensing layer. SPR sensors are often used in such applications as investigation of surface and interface effects, spectroscopy, differential reflectivity, immunoasays. SPR sensors are based on the following principle: when a thin metal layer is illuminated by an incident beam of light, under certain circumstances the energy of the light beam can excite free electrons on the illuminated surface of the metallic film. In particular, the beam will resonate with the surface electrons, which resonance will lead to the creation of an electrical field extending within the range of about 200 nanometers. The resonance occurs at a certain angle of incidence of the incoming light beam and depends on the refractive index of a substance located within the range affected by the generated electrical field. Binding or dissociation of the analyte and an immobilized binding layer at the sensor surface changes the local refractive index at the surface and produces a shift in the resonant angle of incidence, which has been shown to be proportional to the concentration of ligands bonded to an immobilized binding layer up to a predetermined limiting concentration. Thus, by electro-optically monitoring changes of the refractive index at the sensing surface using SPR, qualitative sensing of ligands and quantitative characterization of various binding kinetics and equilibria are possible.
An example of an SPR biosensor is schematically illustrated in FIG. 1. SPR biosensor 2 includes a prism 4 having a test surface thereof coated by a thin metallic film 6. A first type of ligands 8 is immobilized on metallic film 6, and an analyte 10 is introduced into the contacting phase above the test surface. A light source 12 of predetermined wavelength directs an incident beam 14 to metallic film 6, and a photosensitive detector 16 is arranged to monitor the intensity of reflected beam 14xe2x80x2. At a certain angle of incidence xcex1 of beam 14, resonant excitation of electrons (surface plasmons) in metallic film 6 results in absorption of incident beam 14 and, consequently, in an energy loss in the reflected beam 14xe2x80x2, which is observed experimentally as a sharp minimum in the intensity of light received by detector 16, as illustrated in FIG. 2.
While SPR sensors exhibit a high degree of sensitivity to changes in refractive indices, which makes them a useful research tool, immobilizing a binding layer on a metallic layer is both difficult and limiting. It is difficult, because the immobilization technique must attach the ligands in a native conformation and in a uniformly reactive and accessible orientation, to a metallic surface that does not allow for a significant amount of non-specific binding. A number of various immobilization techniques have been described in the art, with the choice of a technique being dependent upon particular ligands involved. Because of these and other difficulties associated with manufacturing SPR sensors, such sensors are expensive. Therefore, it would be desirable to come up with a less expensive device capable of measuring changes of the refractive indices caused by interactions between various ligands.
An example of a suitable device for sensitive and quantitative measurements associated with changes in refractive indices is a critical angle refractometer. The operation of a critical angle refractometer is based on the following principle. When light is incident on a surface separating two media, the light is refracted at the interface between the two media in accordance with Snell""s law:
n Sin I=nxe2x80x2 Sin Ixe2x80x2
where n and nxe2x80x2 are the refractive indices of the two media, and I and Ixe2x80x2 are the angles of incidence and refraction, respectively. Light can always pass from a lower refractive index medium to a higher refractive index medium, because in that case angle Ixe2x80x2 is smaller than angle I. However, when a beam of light passes from an optically denser medium (having a higher index of refraction n) to an optically rarer medium (having a lower index of refraction nxe2x80x2), the angle of refraction Ixe2x80x2 is always greater than the angle of incidence I. As the angle of incidence I increases, the angle of refraction Ixe2x80x2 increases at a faster rate. When Sin I=nxe2x80x2/n, then Sin Ixe2x80x2=1.0 and the angle of refraction Ixe2x80x2=90 degrees. Such an angle of incidence is called the critical angle. When the critical angle condition is met, no light propagates into the optically rarer medium. When the angle of incidence is greater than the critical angle, the light is reflected back into the optically denser mediumxe2x80x94a phenomenon called total internal reflection (T.I.R.). If the separating boundary of the two media is smooth and clean, 100 percent of the incident light is reflected back. The critical angle phenomenon is used for measurements of refractive indices of various fluid or solid materials.
FIG. 3a depicts a critical angle refractometer shown and identified broadly by the reference numeral 22. Refractometer 22 is shown as including a housing 32 having an inclined top surface portion 34 and a horizontal top surface portion 36 adjacent thereto, an LCD display 38 and a keypad input 40 at inclined top surface portion 34. A test assembly 24 is situated on horizontal top surface portion 36. Refractometer 22 is similar to the Leica AR600 automatic refractometer available from Leica Microsystems Inc. The Leica AR600 automatic refractometer is manufactured generally in accordance with the disclosure of commonly-owned U.S. Pat. No. 4,640,616 issued Feb. 3, 1987 and entitled AUTOMATIC REFRACTOMETER. The entire disclosure of U.S. Pat. No. 4,640,616 is incorporated herein by reference as if reprinted in its entirety.
The schematic of FIG. 4 illustrates the opto-electronic measurement system of refractometer 22, which is based on the principles of critical angle refractometry described above. The system comprises a photosensitive linear scanned array (LSA) 44 for providing an output signal as a function of the amount and location of light incident thereon. Linear scanned array 44 includes a plurality of closely adjacent and aligned photoelectric cells 46. The measurement system comprises an optical system for directing light onto linear scanned array 44, wherein the amount and location of light illuminating the LSA depends on the index of refraction of a test sample 51. As shown in FIG. 4, the optical system includes a light source 48 and a prism 50 for receiving light along an optical path 57 from source 48. Prism 50 includes a top surface 54 for receiving test sample 51, a bottom surface 56 parallel to top surface 54 through which light enters and exits the prism, and a pair of internally reflective side surfaces 58 and 60, which define acute included angles with bottom surface 56. A temperature sensor 52 is provided at top surface 54 to read sample temperature for temperature compensation purposes.
Light originating from source 48 travels sequentially through a diffuser 62, a polarizer 64, and a collimating lens 66. The parallel light leaving collimating lens 66 enters an interference filter 68 which transmits essentially monochromatic light at a wavelength of 589 nm. A converging lens 70 is arranged to receive light transmitted by filter 68 and concentrate the light in the direction of a reflecting mirror 72, which is orientated to reflect the light through the bottom surface 56 of prism 50. The light is totally internally reflected by side surface 58 to impinge upon top surface 54. A first portion of light (not shown) incident on top surface 54 at the angles less than the critical angle is refracted into sample 51. A second portion of light 55 incident on surface top 54 at the angles larger than the critical angle is totally internally reflected from top surface 54. Second portion of light 55 is then internally reflected by side surface 60 and exits prism 50 through bottom surface 56. After passing through a lens 73, portion 55 is redirected by a reflecting mirror 74 in the direction of linear scanned array 44. Therefore, light distribution at LSA 44 consists of an illuminated region 47, formed by second portion of light 55, and a non-illuminated region 47a. The boundary between the two regions 47 and 47a is referred to as the shadow line, and its position on linear scanned array 44 is dependent upon the refractive index of test sample 51.
In the Leica AR600 automatic refractometer, the LAS contains almost 2600 individual charge-coupled device (CCD) elements, each of which is a 11 xcexcm2 square. Each CCD, pixel, is capable of converting the intensity of light hitting upon it into an electrical voltage, which is subsequently converted to a digital number between 0 and 255 by supporting circuitry. Each CCD produces a numeric intensity value as an output reading. A typical graph, illustrating illumination intensity from a bare prism (a reference reading of air) as a function of a cell number, is shown in FIG. 5a. The reference reading of air in FIG. 5a is taken by pressing an INITIATE key of keypad input 40 to provide a reference curve 100, corresponding to the illumination distribution at linear scanned array 44 without a sample on top surface 54 of prism 50. When a sample is placed on the prism, the first portion of the light is transmitted through the sample, and second portion of light 55 is reflected toward the LSA, illuminating a part of it, thus, forming a shadow line on the LSA, as described above with regard to FIG. 4. Determination of the shadow line location expressed as the crossover cell number is carried out by a software routine stored in the programmable memory of refractometer 22. During a reading, reference curve 100 is scaled by 94%, as indicated by the dashed curve just below reference curve 100 in FIG. 5b, forming a scaled reference curve 120. The scaling parameter does not have to be 94%, it can vary (80%, 85% for example) to achieve the best precision between consecutive readings. The crossover cell number is then found by a routine, which identifies the cell or cell fraction at which a sample curve 110 intersects with scaled reference curve 120. The crossover cell number is then converted to a refractive index value, based on a calibration reading of a substance of a known refractive index.
Despite the fact that the critical angle reflection phenomenon has been known in the past, there has been no successful effort to bring critical angle refractometers into the analytical art as sensors, capable of detecting and monitoring binding between an analyte and a binding layer having specific affinity to the analyte. Since critical angle refractometers, such as, for example, the above-described Leica AR600 automatic refractometer are inexpensive, compared to commercially available SPR sensors, it would be desirable to use a critical angle refractometer to sense and monitor binding phenomena. Therefore, the need exists to provide a method and device utilizing critical angle refractometry to sense and monitor the presence and the amount of a particular analyte by measuring changes in the refractive index occurring due to specific binding of the analyte to an immobilized binding layer on a sensing surface.
Therefore, it is an object of the present invention to provide a sensing device and method utilizing critical angle refractometry to sense and monitor binding interactions between a sample analyte and a binding layer.
It is another object of the present invention to provide a sensor device, which does not measure changes in a refractive index by using the surface plasmon resonance phenomenon, and thus avoids the need for experimentally rigorous procedure of immobilization of a binding layer on a thin metallic layer. A related object of the invention is to avoid problems associated with oxidation of a metallic layer and the necessity to provide an intermediate layer between the metallic layer and a glass surface in traditional SPR sensors.
It is a further object of the present invention to provide a critical angle based sensor device, which is affordable to manufacture and simple to operate.
It is yet another object of the present invention to provide a critical angle refractometric method and apparatus for measuring changes in the refractive index at a sensing layer by passing light through an optically transparent arrangement to cause the light to be totally internally reflected at the sensing layer.
It is also another object of the present invention to provide a method and device utilizing critical angle refractometry to sense presence or absence of a sample analyte in a contacting phase by measuring the critical angle of total reflection of light at a sensing layer.
It is yet another object of the present invention to provide a critical angle refractometer and method for measuring the rate of a binding reaction between a binding layer and a sample analyte.
In view of these and other objects, an apparatus and method for sensing presence and the amount of an analyte in a contacting phase are provided by using a critical angle refractometer to sense changes in a refractive index of a sensing layer occurring as the interaction between the sample analyte and an immobilized binding layer progresses over time. The apparatus, according to one of the embodiments of the present invention, comprises an automatic critical angle refractometer for obtaining refractive index data with respect to a sample analyte in operative association with an opto-electronic measurement system of the refractometer, and a computer connected for data communication with the refractometer for processing the data and reporting changes in the refractive index as a function of time.
The refractometer measurement system includes a linear scanned array of photosensitive cells, and an optical system for directing light onto the LSA. The light impinging upon the LSA forms a shadow line, dividing the LSA into an illuminated portion and a dark non-illuminated portion. The location of the shadow line is dependent on the refractive index of a binding layer immobilized on the sensing surface. Depending on whether the sample analyte has bonded with the binding layer, the position of the shadow line will change. Therefore, correlation of the shadow line location to a value, which is a function of the refractive index, such as a concentration of the sample analyte in the contacting phase, can be established. The correlation is carried out by software routines stored in the programmable memory of the refractometer.
In accordance with the present invention, a method of using critical angle refractometry for sensing and monitoring interactions between the analyte and the binding layer is provided. An optical system directs light through one or more optically transparent elements to impinge upon the interface between the binding layer and one of the optically transparent elements. The absence or presence of binding between the analyte and the binding layer changes the refractive index of the binding layer. The refractive index of the binding layer, in turn, affects the critical angle of total reflection. The light reflected from the interface at a particular angle impinges on the LSA, creating a shadow line, the location of which can be related to the amount of the analyte bonded to the immobilized binding layer. The same principle enables the method and apparatus of the present invention to monitor and measure the rate of changes in the refractive index, which rate is proportional to the concentration of the analyte in a contacting phase and the strength of affinity between the analyte and the binding layer.
The present invention also provides an apparatus and method for sensing the presence or absence of a particular analyte having specific affinity to the binding layer by measuring changes of the refractive index at the binding layer. Such sensing can be implemented in laboratory tests and home test kits. The method comprises directing a collimated light beam at a particular incident angle through one or more optically transparent elements to impinge upon the interface between the binding layer and one of the optically transparent elements. Depending on whether a particular analyte with specific affinity to the binding layer is present or absent in the contacting phase, the incident angle of light will or will not satisfy the condition for total internal reflection. If the condition for total internal reflection is satisfied, the reflected light will impinge on the LSA or any other sensor capable of detecting light, disposed along the optical path of the reflected light. Therefore, depending on whether the sensor is illuminated by totally internally reflected light, the presence or absence of the analyte can be determined. It is also contemplated that the LSA can be disposed to sense transmitted light, which will illuminate the LSA depending on whether the T.I.R. condition is satisfied. An apparatus for practicing the above-described method comprises a collimated beam of light directed at the interface at a particular angle of incidence. In order to sense the critical angle of total reflection, a single light source capable of moving and changing the angle of incidence is provided. In an alternative embodiment of the apparatus, a plurality of light sources directing light beams at the interface at different angles, are utilized to sense the presence or absence of the analyte. Depending on whether the binding between the analyte and the immobilized binding layer has occurred, the light from one of the light sources becomes totally internally reflected at the interface, therefore, illuminating the light sensor and indicating the presence or absence of the analyte.
In one of the embodiments of the invention, a specialized test assembly allows for operative association between the sample analyte in a contacting phase and the immobilized binding layer. In the preferred embodiment, the apparatus includes a thin, optically transparent element having a selected type of ligands immobilized on an upper surface thereof, forming a binding layer. A flow cell is arranged closely above the transparent element for providing a buffer flow of the contacting phase containing the sample analyte intended for specific binding interaction with the immobilized binding layer. An O-ring or a gasket arranged on the upper surface of the disc is sized to provide a peripheral fluid-tight seal between the binding layer on the sensing surface of the element and the flow cell. A high refractive index coupling liquid is provided between a lower surface of the optically transparent element and the top surface of the refractometer prism. The transparent elements, such as discs, are preferably formed of glass, polystyrene, polycarbonate, or other optically transparent materials with a suitable index of refraction. A particular immobilization technique usually depends in part on the material used to form the disc. By way of example, an antibody, such as an anti-strepavidin antibody, may be immobilized on the upper surface of the optically transparent disc, and its antigen strepavidin introduced in a buffer flow for analysis of binding interactions. By way of further example, with respect to DNA binding protein/DNA ligand interactions, the OccR protein may be immobilized on the upper surface of the optically transparent disc, and its oligonucleotide target introduced in a contacting phase for analysis of binding interactions.
To summarize, the present invention provides a method of using critical angle refractometry for sensing presence or absence of an analyte at a binding layer, the method comprising providing a first optically transparent element and a second optically transparent element, the first optically transparent element having a higher refractive index than that of the second optically transparent element, the second element having the binding layer, providing a contacting phase, allowing the contacting phase to contact the binding layer of the second optically transparent element, passing light through the first and the second optically transparent elements to cause the light to impinge upon an interface between the second optically transparent element and the binding layer, and detecting a location of a boundary between a light area and a dark area on a sensing element, the location of the boundary being indicative of the presence or absence of the ligands at the binding layer. The method further provides a contact layer coupling the first optically transparent element to the second optically transparent element. The contacting phase can be liquid, the second optically transparent element is selected from the group consisting of glass and plastic. The binding layer is selected from the group consisting of carboxymethylated dextran, aldehyde activated dextran, hydrazide activated dextran, silanated surfaces, silanized surfaces, silane, aviden, streptaviden, neutraviden, biotinyl, bifunctional spacer arms, self assembled monolayers, lipids and unchanged or uncoated surface of the second optically transparent element. The contacting phase containing the analyte comprises selecting the analyte from the group consisting of antigens, proteins, glycoproteins, vitamins, microbes, pieces of microbes including bacteria and bacterial fragments, viruses, pieces of viral material, lipids, carbohydrates, toxins, DNA, RNA, DNA and RNA analogs, pathogenic organic molecules, anti-bacterial and anti-viral organic molecules and their analogs, therapeutic agents and drugs.
Another embodiment of the invention is a method of using critical angle refractometry for sensing presence or absence of an analyte at a binding layer of a first optically transparent material, the method comprising providing the first optically transparent material of a higher optical density than that of the binding layer contacting the binding layer with a contacting phase passing light along an optical path through the first optically transparent material to cause the light to impinge upon an interface between the binding layer and the first optically transparent material sensing a boundary between a light area and a dark area on a sensing element disposed along the optical path, and utilizing the location of the boundary to determine the presence or absence of the analyte at the binding layer. The optically transparent material is selected from the group consisting of glass and plastic.
Yet another embodiment of the invention is a method for sensing presence or absence of an analyte at a binding layer, the method comprising providing an interface between the binding layer and an optically transparent element, the interface being located along an optical path, the binding layer and the optically transparent element having different optical densities sufficient to totally internally reflect light impinging on the interface, contacting the binding layer with a contacting phase, illuminating the interface with the light propagating along the optical path, so that a portion of the light totally internally reflected from the interface propagates between the interface and a sensing element disposed along the optical path and illuminates the sensing element to form a light area thereon, and detecting a location of a boundary between the light area and a dark area on the sensing element, the location of the boundary being indicative of the presence or absence of the analyte at the binding layer. The portion of the light propagating between the interface and the sensing element comprises light reflected from the interface or transmitted through the interface.
Another embodiment of the invention is a method of sensing presence or absence of an analyte at a binding layer comprising providing a light beam generated by a light source, providing an interface between the binding layer and an optically transparent element, the binding layer and the optically transparent element having optical densities sufficient to cause the light beam impinging upon the interface to be totally internally reflected, contacting the binding layer with a contacting phase, illuminating the interface by the light beam impinging upon the interface at a predetermined angle of incidence, providing a sensor located at a position in which the sensor can sense the light totally internally reflected at the interface, and sensing the presence or absence of light by the sensor, the presence or absence of light being indicative of the presence or absence of the analyte at the binding layer. The method further comprises altering the predetermined angle of incidence by moving or rotating the light source or by moving or rotating the optically transparent element. The method also comprises providing a plurality of light sources so that altering the angle of incidence is accomplished by illuminating the interface by a light beam from a different light source. The described sensor can comprise a plurality of sensing elements.
And yet another embodiment of the invention is a system for detecting presence or absence of an analyte in a contacting phase, the system comprising an optically transparent element having a binding layer deposited thereon, the binding layer having affinity to the analyte a critical angle refractometer defining an optical path of a collimated light beam impinging upon an interface between the binding layer and the optically transparent element, the contacting phase contacting the binding layer, and a sensor disposed along the optical path to detect changes in an optical density of the binding layer by sensing light travelling along the optical path. The system further comprises a test assembly serving to bring the contacting phase in contact with the binding layer, wherein the optically transparent element is a disposable slide and wherein the contacting phase is a biological fluid. The system further comprises a plurality of light sources, wherein each light source is capable of directing a light beam toward the interface at a predetermined angle of incidence and wherein the sensor comprises a plurality of sensing elements.
The present invention also encompasses a method of monitoring specific binding during a particular reaction involving an analyte, the method comprising immobilizing a binding layer on an optically transparent element; bringing the transparent element into operative association with an opto-electronic measurement system of an automatic critical angle refractometer, introducing a contacting phase containing the analyte to contact the binding layer, using the critical angle refractometer to generate measurement data, including data that are a function of the refractive index of the binding layer, at regular intervals over time, and processing the measurement data to permit analysis of the progress of specific binding of the analyte to the binding layer.